TY - JOUR
T1 - Mechanistic basis for microhomology identification and genome scarring by polymerase theta
AU - Carvajal-Garcia, Juan
AU - Cho, Jang Eun
AU - Carvajal-Garcia, Pablo
AU - Feng, Wanjuan
AU - Wood, Richard D.
AU - Sekelsky, Jeff
AU - Gupta, Gaorav P.
AU - Roberts, Steven A.
AU - Ramsden, Dale A.
N1 - Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/4/14
Y1 - 2020/4/14
N2 - DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.
AB - DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.
KW - BRCA
KW - Chromosome break repair
KW - Microhomology-mediated end joining
KW - Mutational signatures
KW - Pol theta
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U2 - 10.1073/pnas.1921791117
DO - 10.1073/pnas.1921791117
M3 - Article
C2 - 32234782
AN - SCOPUS:85083186652
SN - 0027-8424
VL - 117
SP - 8476
EP - 8485
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 15
ER -